Chemicals

All chemicals used were of A.R. or tissue culture grade, and water for making media and solutions was obtained from Milli Q (Millipore, USA) or Nanopure (Bamstead, USA) purification systems and had a resistivity of at least 18 mega ohms.

2.1.2.1. Serum

Fetal calf serum (FCS) in bottles of 500ml was obtained from Commonwealth Serum Laboratories, (Melbourne, Australia). Prior to use the FCS was heat inactivated. After sterilization the serum was stored at -20°C in 50ml sterile plastic flasks (Coming, USA) each containing 30ml of serum. The suitability of the serum

was tested by culturing day-2 mouse embryos to the blastocyst stage in medium supplemented with a sample from the batch under test.

Sheep serum was obtained by jugular venipuncture using a 20ml disposable plastic syringe fitted with a disposable 18g needle (Terumo, Japan). Blood was gently emptied into 100ml sterile plastic specimen containers which were then placed at an angle and allowed to clot overnight. The following day the serum was separated by centrifugation, heat inactivated, filtered sterile and stored at -20°C in 5ml portions in 6ml sterile plastic tubes (Falcon Plastics, BD, USA).

2.7.2.2. Media for Culture and Handling of Embryos

Whitten's medium (WM) (Whitten, 1971) prepared according to the modifications of Cross and Brinster (1973), was used for culture of mouse embryos. The modifications included a reduction in the concentration of sodium pyruvate from 0.318mM to 0.270mM and of calcium lactate from 1.72mM to 0.860mM and the inclusion of 10.8uM EDTA (Abramczuk, Solter and Koprowski, 1977). Hepes buffered Whitten's medium (HWM) containing 3.619mM sodium bicarbonate and 25mM sodium Hepes adjusted to pH 7.2 to 7.25 at room temperature was used for handling mouse embryos. Both the media were filtered sterile using disposable filter units of 0.2um pore size (Nalgene, USA) and stored at 4°C in 50ml sterile flasks (Coming,US A). Fresh media (WM and HWM) were prepared every three to four weeks.

Synthetic oviductal fluid (SOF) (Tervit, Whittingham and Rowson, 1972) was used for culture of sheep embryos and was prepared according to the modifications of Walker, Seamark, Quinn, Wames, Ashman, Smith and Ancell (1988,1990). Hepes buffered SOF (HSOF) was used for handling and temporary holding of sheep embryos. This medium was prepared according to the modifications of Walker et al. (1990). It contained 4.0 ImM sodium bicarbonate and 25mM sodium Hepes and the pH was adjusted to 7.5. The working SOF and HSOF contained 20% heat inactivated sheep serum. The media were treated as described for WM and HWM but fresh SOF and HSOF were prepared every fortnight.

Hepes buffered medium 199 was used for flushing sheep uterine horns in the recovery of embryos. Hepes buffered powdered medium 199 was obtained from Flow laboratories (Irvine, Scotland) and was made up in purified water according to the instructions of the manufacturer. Sodium bicarbonate in the order of

l0.012mM was incorporated. The medium was filtered sterile and stored at 4°C. Prior to use FCS 5% (v/v) was incorporated in the medium.

2.1.3. Sterile Plasticware

Gamma irradiated sterile tissue culture grade plasticware was obtained from Nunc, Denmark; Coming, USA; and Falcon Plastics, Becton Dickinson, USA. Sterile disposable filter assemblies (110ml, 250ml and 1L) were obtained from Nalgene, USA. Sterile disposable filters were obtained from Sartorius, Germany and Millipore, USA. Sterile disposable hypodermic needles and plastic syringes were obtained from Terumo Corporation, Japan, and Becton and Dickinson, USA.

2.1.4. Hormones

Intravaginal sponges (Chrono-Gest, Intervet Australia Pty, Artarmon, NSW) containing 40mg flugestone acetate were used for synchronization of oestrous in donor and recipient ewes. Hormone preparations used for the superovulation of mice were pregnant mare serum gonadotropin(PMSG; Folligon, Intervet International B.V., Boxmeer, Holland) and human chorionic gonadotropin (hCG, Chorulon, Intervet International B.V., Boxmeer, Holland). The hormone preparations used for sheep superovulation were PMSG (Folligon) and follicle stimulating hormone (FSH; F.S.H-P, Schering Corporation, USA).

2.2. Methods

2.2.7. Sterile Techniques

Unless otherwise stated all techniques and procedures described in this report were carried out observing standard sterile or aseptic procedures.

2.2.2. Generation of Embryos

2.2.2.1. Synchronization of Mouse Oestrous Cycles by Whitten Effect

A male was placed in close proximity with 10 to 20 Swiss outbred (S.O.) females separated by a wire grid to synchronise the oestrous cycles of the females. This is termed the Whitten effect (Whitten, 1957b). On third night of exposure to the "teaser" male, the females were placed with S.O. stud males in the ratio of one

male to one female. The following morning the females were checked for the presence of copulation plugs in the vagina. The day copulation was detected was considered as day 1 of pregnancy.

2.22.2. Superovulation of Mice

When a large number of preimplantation embryos were required S.O. females were treated with gonadotropins prior to mating (Fowler and Edwards, 1957). Superovulation was induced by the administration of 10 i.u. of pregnant mare serum gonadotropin (PMSG) intraperitoneally. Immediately after PMSG injection the females were exposed to the "teaser" male as described in section 2.2.2.1. Forty six to forty eight hours later 10 i.u. of human chorionic gonadotropin (hCG) was administered intraperitoneally. After hCG administration each female was placed in a cage with a stud male in a ratio of one female to one male and checked for copulation plug the following morning. PMSG and hCG were made up in sterile PBS to contain 100 i.u. per ml for each preparation and stored at -20°C.

2.22.3. Sup er ovulation and Insemination of Ewes

Mature Australian Merino ewes were used as embryo donors. Their oestrous cycles were synchronised by the insertion of intravaginal sponges containing 40mg flugestone acetate (Chrono-Gest, Intervet Australia Pty, Artarmon, NSW) for 12 days. Superovulation was induced with follicle stimulating hormone (FSH) and pregnant mare serum gonadotropin (PMSG) in combination (Ryan, Bilton, Hunton and Maxwell, 1984). Treatment consisted of 12 mg FSH and 500 i.u. of PMSG given intramuscularly two days before removal of the sponges.

The ewes were joined with rams fitted with sire-sine harnesses and crayons in the ratio of one ram to two ewes, immediately after removal of the sponge. To ensure fertilization intrauterine insemination was carried out by laparoscopy 36-48 hours after sponge removal (Killeen and Caffery, 1982). The ewes were starved for 12 hours prior to laparoscopy. Each ewe was placed on the laparotomy cradle (Blockey, Englund and Cumming, 1972) and the wool on the abdomen was shorn over an area extending about 20cm anterior to the mammary glands. After injection of local anaesthetic two cannulae (10.5 and 7.0mm O.D.) were inserted with the aid of trocars into the abdomen about 5 to 8cm anterior to the udder on either side 3 to 5 cm from the midline. Care was taken not to damage the large subcutaneous blood vessels. The trocar was directed so as not to puncture the

bladder or damage the femoral artery. After inflation of the abdominal cavity with CC>2 an endoscope (American Cytoscope Makers Inc., New York, USA) (9mm O.D.) was introduced through the larger cannula. In ewes on which laparotomy had not been performed previously and which were in medium or less than medium condition, the uterine horns were usually visible and accessible immediately after insertion of the endoscope. Adhesions caused by previous operations or more than normal amounts of body fat sometimes hindered the operation and made access to the uterus more difficult. In such cases the intestines and connective tissues were manipulated with a probe.

Semen was collected from Merino rams by electroejaculation and the motility and density of the ejaculates were determined under a microscope. The specimen was again observed under the microscope after dilution 1:1 with PBS and after completion of insemination to determine quality of the semen. Inseminations were performed with a glass insemination pipette 25cm in length (I.D. 2.0mm, O.D. 6.5mm) to which a 1ml syringe was fitted. The other end of the pipette which was tapered and sharp was introduced into the abdominal cavity through the smaller cannula and directed to the uterus. After puncture of the uterine wall semen was expelled from the pipette. The volume of the inseminate was about 0.05ml.